The Saccharomyces Cerevisiae Rtg2 Gene Is a Regulator of Aconitase Expression under Catabolite Repression Conditions

The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACO1 mRNA level is further reduced when glutamate is supplied with glucose. To further explore regulation of ACO1 expression, we have screened for mutations that reduce expression of an ACO1-lacZ fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACO1 only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient''s muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1^R182H mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ?yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

Isolation of coding sequences from bovine cosmids by means of exon trapping

Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and β-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes. Received: 23 November 1996 / Accepted: 3 March 1997     

Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, 11778, or 14484 Leber's hereditary optic neuropathy mitochondrial DNA mutation

Leber's hereditary optic neuropathy (LHON) is a form of blindness caused by mitochondrial DNA (mtDNA) mutations in complex I genes. We report an extensive biochemical analysis of the mitochondrial defects in lymphoblasts and transmitochondrial cybrids harboring the three most common LHON mutations: 3460A, 11778A, and 14484C. Respiration studies revealed that the 3460A mutation reduced the maximal respiration rate 20-28%, the 11778A mutation 30-36%, and the 14484C mutation 10-15%. The respiration defects of the 3460A and 11778A mutations transferred in cybrid experiments linking these defects to the mtDNA. Complex I enzymatic assays revealed that the 3460A mutation resulted in a 79% reduction in specific activity and the 11778A mutation resulted in a 20% reduction, while the 14484C mutation did not affect the complex I activity. The enzyme defect of the 3460A mutation transferred with the mtDNA in cybrids. Overall, these data support the conclusion that the 3460A and 11778A mutants result in complex I defects and that the 14484C mutation causes a much milder biochemical defect. These studies represent the first direct comparison of oxidative phosphorylation defects among all of the primary LHON mtDNA mutations, thus permitting insight into the underlying pathophysiological mechanism of the disease.     

Mutation frequency is reduced in the cerebellum of Big Blue mice overexpressing a human wild type SOD1 gene

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by motor neuron degeneration. A similar disease phenotype is observed in mice overexpressing a mutant human hSOD1 gene (G93A, 1Gurd(1)). Mice transgenic for lacI (Big Blue) and human mutant (1Gurd(1), Mut hSOD1) or wild type (2Gur, Wt hSOD1) SOD1 genes were used to examine spontaneous mutation, oxidative DNA damage, and neurodegeneration in vivo. The frequency and pattern of spontaneous mutation were determined for forebrain (90% glia), cerebellum (90% neurons) and thymus from 5-month-old male mice. Mutation frequency is not elevated significantly and mutation pattern is unaltered in Mut hSOD1 mice compared to control mice. Mutation frequency is reduced significantly in the cerebellum of Wt hSOD1 mice (1.6x10(-5); P=0.0093; Fisher's Exact Test) compared to mice without a human transgene (2.7x10(-5)). Mutation pattern is unaltered. This first report of an endogenous factor that can reduce in vivo, the frequency of spontaneous mutation suggests potential strategies for lowering mutagenesis related to aging, neurodegeneration, and carcinogenesis.     

Exome sequencing identifies compound heterozygous mutations in CYP4V2 in a pedigree with retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of progressive retinal degenerations characterized by pigmentation and atrophy in the mid-periphery of the retina. Twenty two subjects from a four-generation Chinese family with RP and thin cornea, congenital cataract and high myopia is reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family presented with bone spicule-shaped pigment deposits in retina, retinal vascular attenuation, retinal and choroidal dystrophy, as well as punctate opacity of the lens, reduced cornea thickness and high myopia. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. After mutation analysis in a few known RP candidate genes, exome sequencing was used to analyze the exomes of 3 patients III2, III4, III6 and the unaffected mother II2. A total of 34,693 variations shared by 3 patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in the rest family members by PCR and Sanger sequencing. Compound heterozygous c.802-8_810del17insGC and c.1091-2A>G mutations of the CYP4V2 gene, known as genetic defects for Bietti crystalline corneoretinal dystrophy, were identified as causative mutations for RP of this family.     

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.     

Myelocystocele-cloacal exstrophy in a pedigree with a mitochondrial 12S rRNA mutation, aminoglycoside-induced deafness, pigmentary disturbances, and spinal anomalies

A large Filipino-American family with progressive matrilineal hearing loss, premature graying, depigmented patches, and digital anomalies was ascertained through a survey of a spina bifida clinic for neural crest disorders. Deafness followed a matrilineal pattern of inheritance and was associated with the A1555G mutation in the 12S rRNA gene (MTRNR1) in affected individuals as well as unaffected maternal relatives. Several other malformations were found in carriers of the mutation. The proband had a myelocystocele, Arnold-Chiari type I malformation, cloacal exstrophy, and severe early-onset hearing loss. Several family members had premature graying, white forelock, congenital leukoderma with or without telecanthus, somewhat suggestive of a Waardenburg syndrome variant. In addition to the patient with myelocystocele, two individuals had scoliosis and one had segmentation defects of spinal vertebrae. The syndromic characteristics reported here are novel for the mitochondrial A1555G substitution, and may result from dysfunction of mitochondrial genes during early development. However, the mitochondrial A1555G mutation is only rarely associated with neural tube defects as it was not found in a screen of 218 additional individuals with spina bifida, four of whom had congenital hearing loss.     

Altered Mitochondrial Function in Canine Ceroid-Lipofuscinosis

The neuronal ceroid-lipofuscinoses (NCL) are a group of autosomal recessively inherited neurodegenerative disorders characterized by progressive dementia, neuronal atrophy, and premature death. The late infantile and juvenile types of NCL show massive accumulation of mitochondrial ATP synthase subunit c protein in both mitochondria and lysosomes. The specific accumulation of this mitochondrial protein suggests that mitochondrial function may be impaired in the NCL diseases. Therefore, a study was conducted to determine whether oxidative phosphorylation is altered in liver mitochondria from English setters with NCL, an animal model in which there is also massive accumulation of the subunit c protein. The ADP/O ratios were significantly depressed in affected and carrier dogs, suggesting that the disease mutation led to a partial uncoupling of oxidative phosphorylation. On the other hand, ADP-stimulated respiration rates were higher than normal in both carriers and affected dogs. The increased respiration rates were highest in the carriers, and may reflect a compensatory response to the reduced efficiency of oxidative phosphorylation. Accompanying the increased respiration rates were elevations in mitochondrial ADP content with the elevation being greater in the carriers than in the affected dogs. This suggests that the increased respiration rates may be due, at least in part, to enhanced ADP uptake by the mitochondria. In the carriers, the enhanced respiration rate may be sufficient to offset the reduced efficiency of oxidative phosphorylation. In the affected animals, which had lower respiration rates than the carriers, the enhanced respiration rates may not be sufficient to offset the reduced efficiency of oxidative phosphorylation. Impaired mitochondrial function may therefore contribute to the disease pathology.     

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation

Iron?sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron?sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5′-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.     

Mutations in KCNJ13 cause autosomal-dominant snowflake vitreoretinal degeneration

Snowflake vitreoretinal degeneration (SVD, MIM 193230) is a developmental and progressive hereditary eye disorder that affects multiple tissues within the eye. Diagnostic features of SVD include fibrillar degeneration of the vitreous humor, early-onset cataract, minute crystalline deposits in the neurosensory retina, and retinal detachment. A genome-wide scan previously localized the genetic locus for SVD to a 20 Mb region flanked by D2S2158 and D2S2202. This region contains 59 genes, of which 20 were sequenced, disclosing a heterozygous mutation (484C > T, R162W) in KCNJ13, member 13 of subfamily J of the potassium inwardly rectifying channel family in all affected individuals. The mutation in KCNJ13, the gene encoding Kir7.1, was not present in unaffected family members and 210 control individuals. Kir7.1 localized to human retina and retinal pigment epithelium and was especially prevalent in the internal limiting membrane adjacent to the vitreous body. Molecular modeling of this mutation predicted disruption of the structure of the potassium channel in the closed state located immediately adjacent to the cell-membrane inner boundary. Functionally, unlike wild-type Kir7.1 whose overexpression in CHO-K1 cells line produces highly selective potassium current, overexpression of R162W mutant Kir7.1 produces a nonselective cation current that depolarizes transfected cells and increases their fragility. These results indicate that the KCNJ13 R162W mutation can cause SVD and further show that vitreoretinal degeneration can arise through mutations in genes whose products are not structural components of the vitreous.     

In vitro phosphorylation of bovine cardiac muscle high molecular weight calmodulin binding protein by cyclic AMP-dependent protein kinase and dephosphorylation by calmodulin-dependent phosphatase

MERRF (myoclonic epilepsy with ragged-red fibers) is a severe, multisystem disorder characterized by myoclonus, seizures, progressive cerebellar syndrome, muscle weakness, and the presence of ragged-red fibers in the muscle biopsy. MERRF is associated with heteroplasmic point mutations, either A8344G or T8356C, in the gene encoding the mitochondrial tRNALys. The human ro cell system was utilized to examine the phenotypic consequences of these mutations, and to investigate their molecular genetic causes. Wild-type and mutant transmitochondrial cell lines harboring a pathogenic point mutation at either A8344G or T8356C in the human mitochondrial tRNALys gene were isolated and examined. Mitochondrial transformants containing 100% mutated mitochondrial DNAs (mtDNAs) exhibited severe defects in respiratory chain activity, in the rates of protein synthesis, and in the steady-state levels of mitochondrial translation products as compared with mitochondrial transformants containing 100% wild-type mtDNAs. In addition, both mutant cell lines exhibited the presence of aberrant mitochondrial translation products. These results demonstrate that two different mtDNA point mutations in tRNALys result in fundamentally identical defects at the cellular level, and that these specific protein synthesis abnormalities contribute to the pathogenesis of MERRF. (Mol Cell Biochem 174: 215–219, 1997)     

Cloning and characterization of rat neuronal apoptosis inhibitory protein cDNA

The human neuronal apoptosis inhibitory protein (NAIP) gene was originally discovered because of its deletion in infantile spinal muscular atrophy (SMA), a childhood genetic disorder characterized by motor neuron loss and progressive paralysis with muscular atrophy. Although SMA is now known to be caused by deletions of survival motor neuron (SMN), the fact that NAIP is an anti-apoptotic protein is consistent with the NAIP gene modifying SMA severity. Here we report the cloning of a 1.5 kb rat NAIP cDNA fragment which contains BIR-3 (third baculovirus inhibitory repeat) domain. This fragment shows 78% homology to the human NAIP and 86% homology to the murine counterpart. We have investigated the distribution of NAIP mRNA expressing neurons by in situ RT-PCR technique in the rat central nervous system (CNS). Although all of the neurons appeared to express NAIP mRNA ubiquitously, pronounced elevation of NAIP mRNA expression was observed in the areas innervated by glutamatergic neurons after kainic acid (KA) injection. We have raised an anti-rat NAIP antiserum in rabbits using NAIP cDNA and recombinant rat NAIP, and carried out an immunohistological investigation. We observed highly immunoreactive neuronal subpopulations in the retinal ganglion, cerebral cortex, hippocampus, basal forebrain, thalamus, areas of midbrain, Purkinje cells of the cerebellum, and motor neurons in the spinal cord. Increased immunoreactivity of glutamatergic neurons was also observed broadly in the CNS after KA treatment. This study provides additional evidence that expression of mRNA and gene products of NAIP seem to be regulated in response to excessive stimuli or injuries in rat CNS, and these results are compatible with an anti-apoptotic role of NAIP in acute SMA as well as in brain injuries.     

Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain

Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.     

A New Mouse Allele of Glutamate Receptor Delta 2 with Cerebellar Atrophy and Progressive Ataxia

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)–PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.     

Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.